Cholesterol and its fatty acid esters are important compounds for human beings as they are components of nerve and brain cells and are precursors of other biological materials, such as bile acid and steroid hormones (P. L. Yeagle, Biology of Cholesterol, CRC Press: Its function and metabolism in biology and medicine Plenum: New York, 1972). Cholesterol determination in blood is clinically important for the diagnosis of heart diseases since accumulation of cholesterol and its fatty acid esters in blood due to excessive ingestion can be fatal (D. Noble, Anal. Chem., 1993, 65, 1037A-41A). The normal range of blood serum values extends from 3 to 6 mm for total cholesterol while in the hyperlipidamic condition the level can increases to 10 mM It is therefore desired to develop techniques that allow convenient and rapid determination of cholesterol.
Various methods have been employed in the art for stabilization and immobilization of enzymes within carbon paste or covalently liking it to the surface of glassy carbon electrode or immobilizing it within a polymer film for the preparation of enzyme electrode. In recent years, enzyme immobilization with the retention of enzyme activity within a sol-gel matrix has become a potential tool for development of new biosensors. Avnir et al disclose the immobilization of organic compounds in inorganic supports by introducing the organic compound with a polymerization precursor [J. Phys. Chem., 88 (1984), 5969]. Sol-gel processed materials are known for their use in development of ceramic films for conductive, optical, mechanical and electro-optic applications [Brinker, C. J., and Scherer, G. W., Sol-Gel Science, Academic Press, New York, (1989), Klein, L. C., Annu. Rev. Mater. Sci., 23 (1993) 437]. Braun et al report that alkaline phosphatase retains its activity when immobilized in a sol-gel matrix [Mater. Lett., 10 (1990) 1]. There is disclosure in the art of the immobilization of enzymes including glucose oxidase within a sol-gel matrix [Yamanaka et al, Chem. Mater. 4 (1992) 495; Shtelzer et al, Biochem. Biotechnol., 19 (1994) 293; Narang et al, Anal. Chem., 66 (1994) 3139].
Audebert and Sanchez report the construction of a ferrocene mediated sol-gel biosensor using a two stage sol-gel preparation method based on TMOS and commercial colloidal silica of varying particle size [Chem. Mater. 5 (1993) 911]. According to this literature reference, more than 80% of the glucose oxidase retains its activity in the gel and the Faradic response of the electrode agrees with theoretical calculations based on this activity. Lev et al disclose the use of sol-gel derived composite silica-carbon electrodes and claim the dual advantage of both the porosity and rigidity of the silica matrix and the electrical conductivity of the graphite [Anal. Chem., 66 (1994) 1747]. In this disclosure, glucose oxidase is first adsorbed on the surface of the carbon powder and then used for the preparation of the sol-gel film on a glassy carbon electrode. Kurokawa et al report a similar method where fabricated glucose oxidase doped sol-gel composite is made of various composite fibers such as cellulose or titanium propoxide [Biotechnol. Bioeng., 42 (1993) 394; Biotechnology 7 (1993) 5].
The co-immobilisation of cholesterol oxidase and horse radish peroxidase in a sol gel film is disclosed for example in Analytica Chimica Acta Vol 414, 23 pp, 2000, the method of this disclosure comprises physical adsorption, physically entrapped sandwich and the use of microencapsulation technique for the immobilization of cholesterol and horse radish peroxidase on tetra ortho silicate derived sol gel films. The response time for cholesterol estimation is more than 100 minutes. A response time of 50 seconds was observed amperometrically with a physically entrapped enzyme sandwich sol gel film. Further the enzyme electrode is reported to be stable for a period of 8 weeks only.
Biosensors used in the art suffer from several drawbacks in terms of stability and shorter shelf life. Several have reported methods of immobilization of biorecognition elements for use in chemical sensing researchers [R. F. Taylor, Protein Immobilizing Fundamentals and Applications: Marcel Dicker, New York (1975) Chapter 8, 263-303 and H. H. Weetall, Immobilized Enzyme; Antigen, Antibodies and Peptides Preparation and Characterization: Marcel Dicker, New York (1975) Chapter 6, 263-303]. The methods reported in literature can generally be classified into one of the following categories (1) physisorption (2) covalent attachment or (3) entrapment, among which physisorption is the simplest immobilization approach.
Several disadvantages arise with these methods of immobilization such as problems associated with the large size of the biorecognition elements (e.g. proteins and enzymes). Physisorption produces a range of biorecognition element orientations and apparent biding affinities. Besides physisorption generally leads to a population of biorecognizing elements that is completely unresponsive to target analyte. The immobilized species is completely unresponsive to target analyte. The immobilized species will often leach/desorb from sensing interface because there are no covalent bonds. Covalent schemes generally lead to more stable and uniform (interim of biorecognition orientation) interface and enzyme leaching is minimized. Unfortunately covalent attachment can involve one or more chemical transformation and tends to be time consuming and can be costly.
U.S. Pat. No. 6,342,364 provides a sensor that electrochemically determines cholesterol in low density lipoprotein by only one feed of a sample. The sensor has: an electrode system that is mounted on an electrically insulating base plate and includes at least a working electrode and a counter electrode; an enzyme layer formed on the base plate with the electrode system; and a reagent layer that is arranged before the enzyme layer in a sample solution supply path to the electrode system. The enzyme layer includes at least an oxidoreductase and an electron mediator. The reagent layer includes a reagent that depresses reactivity of cholesterol in lipoproteins other than the low density lipoprotein with the oxidoreductase, for example, a reagent that attaches to lipoproteins other than the low density lipoprotein to form a water-soluble complex. However, the shelf life of this sensor is too low.
U.S. Pat. No. 6,214,612 discloses a cholesterol sensor for quantitative determination of cholesterol is provided containing an electrode system and a reaction reagent system. The electrode system contains a measuring electrode such as a carbon electrode and a counter electrode, and the reaction reagent system contains cholesterol dehydrogenase, nicotinamide adenine dinucleotide and an oxidized electron mediator. Electron mediators include ferricyanide, 1,2-naphthoquinone-4-sulfonate, 2,6-dichlorophenol indophenol, dimethylbenzoquinone, 1-methoxy-5-methylphenazinium sulfate, methylene blue, gallocyanine, thionine, phenazine methosulfate and Meldola's blue. Diaphorase, cholesterol esterase and a surfactant may also be present. The electrode system is on an insulating base plate, and the base plate has a covering member containing a groove that is a sample supplying channel which extends from an end of the base plate to the electrode system. A reaction layer containing the reagent system in dry form and a layer of a hydrophilic polymer is provided on the base plate or the covering member, or on both the electrode system and covering member so as to be exposed to the sample supplying channel. During operation, the electron mediator is reduced in conjunction with oxidation of cholesterol in a sample by cholesterol dehydrogenase, and an amount of current required to electrochemically re-oxidize the electron mediator is directly proportional to a quantity of cholesterol present in the sample. However, the sensor has a low shelf life and also potentially shows leaching of both the mediator and the enzyme.
U.S. Pat. No. 6,071,392 discloses a cholesterol sensor which comprises comprises an electrode system having a measuring electrode and a counter electrode formed on an electrically insulating base plate, an electrode coating layer for covering the electrode system and a reaction reagent layer formed on or in the vicinity of the electrode coating layer, wherein the reaction reagent layer comprises at least an enzyme for catalyzing cholesterol oxidation, an enzyme having a cholesterol ester hydrolyzing activity and a surfactant, the electrode coating layer comprises at least one selected from the group consisting of water-soluble cellulose derivatives and saccharides and is contained at such a concentration that imparts sufficient viscosity to a sample solution for enabling it to hinder invasion of said surfactant into said electrode system when said electrode coating layer is dissolved in said sample solution supplied to said sensor. The sensor of this patent is aimed at eliminating impairment of sensor response due to electrode degeneration caused by invading surfactant into the electrode system. While the response time is stated to be low, the shelf life is again not high due to potential enzymatic leaching.
U.S. Pat. No. 6,117,289 discloses a cholesterol sensor which comprises an electrode system composed of at least a measuring electrode and a counter electrode and disposed on an electrically insulating base plate and a reaction layer formed on or in the vicinity of the electrode system. The reaction layer contains cholesterol esterase for catalyzing the conversion of cholesterol ester into cholesterol, cholesterol oxidase and a surfactant. The response time was up to nine minutes. Additionally the presence of a surfactant can result in electrode degradation.
Electrochemically polymerised conducting polymers have also received considerable attention over the last two decades. The remarkable switching capacity of these materials between the conducting oxidised (doped) and the insulating reduced (undoped) state is the basis of many applications. For example, polyconjugated conducting polymers have been proposed for biosensing applications because of advantageous characteristics such as direct and easy deposition on the sensor electrode by electrochemical oxidation of monomer, control of thickness by deposition of charge and redox conductivity and polyelectrolyte characteristics of the polymer useful for sensor applications.
It is therefore highly desirable to develop biosensors that allow conventional and rapid determination of cholesterol.